The product of the p53 gene suppresses cell growth and plays a critical role in suppressing development of human tumors. Wild-type (wt) p53 and its mutant form have been analyzed in an in vivo system in which the inducible expression of wt-p53 prevents cells from entering the S phase of the cell cycle. The growth arrest occurs near the restriction point in late G1 phase of the cell cycle and the expression of b-Myb, PCNA and DNA polymerase alpha mRNAs was significantly repressed. Biochemical analyses indicated that the antiproliferative effect exerted by the wt-p53 correlates with the presence of a unique conformational state characterized by increased phosphorylation. To identify which p53 residues are phosphorylated, we examined DNA-PK's ability to phosphorylate synthetic peptides of the human p53 protein. Serines at positions 15 and 37 in the amino terminal domain of human p53 were phosphorylated. To prevent phosphorylation at these sites, mutants were constructed that changed the codons for Ser-15 or Ser-37 to alanine codons. Expression of p53 - Ala-37 blocked progression of the cells into S phase; however, p53- Ala-15 was partially defective in blocking cell cycle progression and caused a significant reduction in the steady-state level of p53. 2-D phosphopeptide mapping showed that serines at positions 15, 9 and 33 in the amino terminal domain of human wt-p53 protein are phosphorylated in vivo. In two natural mutants from human tumors, phosphorylation at Ser-15 was reduced compared to the wt-p53. No changes were observed at the other sites. We suggest that phosphorylation of p53 at a specific site may contribute to the activity of wt-p53 to block cell growth.